The long-term objective of this proposal is to delineate mechanisms of the beneficial effects of growth hormone deficiency on mitochondrial function, stress resistance and health span. Our research has been focused on understanding the hypothesis that in long living animals, an upregulation of thiol metabolism leads to greater protection from cellular stress. The applicant's work has established that growth hormone (GH) and IGF-I are major players in longevity assurance. Mice with hereditary dwarfism (Ames) and those that lack a functional GH receptor (GHRKO) exhibit GH deficiency or resistance (respectively), delayed aging, and enhanced stress resistance. The global hypothesis to be tested is that thiol metabolism plays a key role in aging and that GH modulates key components of this pathway ultimately leading to changes in health span (via stress resistance/protection) and lifespan. Thus, reduced GH signaling confers a biologic advantage to dwarf mice leading to better scavenging of toxic metabolic byproducts, altered mitochondrial function and enhanced longevity. To further address and define this global hypothesis, two working hypotheses will be tested in this proposal, both of which focus on the relationship between GH, thiol metabolism and aging. The first is that the susceptibility of mitochondria to molecular insults is controlled by mechanisms that involve GH and thiol metabolism including protein S-thiolation and gluta- thione S-transferase (GST) expression. As such, increased mitochondrial GSH/GSSG leads to increased glutathionylation of respiratory chain complexes. This protein modification renders these proteins more resistant to ROS-induced proteolytic degradation suggesting a protective role of glutathionylation and represents a key mechanism of cellular stress resistance. Some of the GSTs (key for detoxification) are regulated by GH and expression levels are indicative of disease susceptibility but little is known about the relationship between GH, GST and aging. The second hypothesis is that thiol metabolism and DNA methylation patterns are determined by circulating GH levels and dietary methionine (MET). The MET metabolic pathway is highly upregulated in Ames mice resulting in increased GSH and differential DNA methylation. In this project, the applicant plans to elucidate the relationship between GH, thiol metabolism and cellular protection by: 1) directly linking the enhanced respiratory and antioxidative activities in dwarf mice to increased mitochondrial GSH and glutathionylation of these proteins; 2) providing direct evidence that the lack of GH is responsible for substrate-specific enhancement of the GST system; 3) defining the changes in thiol metabolism linked to stress resistance and longevity following altered dietary MET; and 4) establishing the first epigenomic profile of a long-living mouse. Determining GH-dependent pathways and mechanisms may suggest therapeutic interventions to enhance stress resistance, delay aging, treat aging-related disorders and extend health span in humans.